Receptor-like Tyrosine Kinase Research Articles

Delving into the Role of Receptor-like Tyrosine Kinase (RYK) in Cancer: In Silico Insights into Its Diagnostic and Prognostic Utility

The RYK gene encodes a receptor-like tyrosine kinase crucial for several biological processes, including development, tissue homeostasis, and cancer. This study utilized data from the Cancer Genome Atlas Project (TCGA) to evaluate RYK expression at both mRNA and protein levels in various cancers, determine its prognostic significance, and explore its involvement in cancer-related signaling pathways. Elevated levels of RYK mRNA were identified in cholangiocarcinoma (CHOL), pancreatic adenocarcinoma (PAAD), glioblastoma multiforme (GBM), lung squamous cell carcinoma (LUSC), brain lower grade glioma (LGG), head and neck squamous cell carcinoma (HNSC), liver hepatocellular carcinoma (LICH), esophageal carcinoma (ESCA), and colon adenocarcinoma (COAD), while RYK protein levels were observed to be increased in colon adenocarcinoma (COAD), GBM, LICH, cervical and endocervical adenocarcinoma (CESC), and breast invasive carcinoma (BRCA). Additionally, RYK overexpression correlated with poorer prognosis in several cancers, including PAAD, LICH, BRCA, ESCA, COAD, and CESC. Furthermore, RYK showed a positive correlation with the upregulation of multiple receptors and coreceptors in the WNT signaling pathway in various types of cancer. In terms of cancer-related signaling pathways, RYK was found to potentially interact with DNA damage, TSC/mTOR, PI3K/AKT, EMT, RTK, RAS/MAPK, ER hormone, AR hormone, and the cell cycle. This study provides new and previously unreported insights into the role of RYK in cancer biology.

Progranulin and EGFR modulate receptor-like tyrosine kinase sorting and stability in mesothelioma cells.

Progranulin is a growth factor with pro-tumorigenic activity. We recently demonstrated that in mesothelioma, progranulin regulates cell migration, invasion, adhesion, and in vivo tumor formation by modulating a complex signaling network involving multiple receptor tyrosine kinase (RTK)s. Progranulin biological activity relies on epidermal growth factor receptor (EGFR) and receptor-like tyrosine kinase (RYK), a co-receptor of the Wnt signaling pathway, which are both required for progranulin-induced downstream signaling. However, the molecular mechanism regulating the functional interaction among progranulin, EGFR, and RYK are not known. In this study, we demonstrated that progranulin directly interacted with RYK by specific enzyme-linked immunosorbent assay (ELISA) (KD = 0.67). Using immunofluorescence and proximity ligation assay, we further discovered that progranulin and RYK colocalized in mesothelioma cells in distinct vesicular compartments. Notably, progranulin-dependent downstream signaling was sensitive to endocytosis inhibitors, suggesting that it could depend on RYK or EGFR internalization. We discovered that progranulin promoted RYK ubiquitination and endocytosis preferentially through caveolin-1-enriched pathways, and modulated RYK stability. Interestingly, we also showed that in mesothelioma cells, RYK complexes with the EGFR, contributing to the regulation of RYK stability. Collectively, our results suggest a complex regulation of RYK trafficking/activity in mesothelioma cells, a process that is concurrently regulated by exogenous soluble progranulin and EGFR. NEW & NOTEWORTHY The growth factor progranulin has pro-tumorigenic activity. In mesothelioma, progranulin signaling is mediated by EGFR and RYK, a co-receptor of the Wnt signaling. However, the molecular mechanisms regulating progranulin action are not well defined. Here, we demonstrated that progranulin binds RYK and regulates its ubiquitination, internalization, and trafficking. We also uncovered a role for EGFR in modulating RYK stability. Overall, these results highlight a complex modulation of RYK activity by progranulin and EGFR in mesothelioma.

PAX7, PAX9 and RYK Expression in Cleft Affected Tissue.

Background and Objectives: Cleft lip with or without cleft palate is one of the most common types of congenital malformations. Transcription factors paired box 7 and 9 (PAX7, PAX9) and receptor-like tyrosine kinase (RYK) have been previously associated with the formation of orofacial clefts but their exact possible involvement and interactions in the tissue of specific cleft types remains uncertain. There is a limited number of morphological studies analyzing these specific factors in cleft affected tissue due to ethical aspects and the limited amount of available tissue material. This study analyses the presence of PAX7, PAX9, and RYK immunopositive structures within different cleft affected tissue to assess their possible involvement in cleft morphopathogenesis. Materials and Methods: Cleft affected tissue was collected from non-syndromic orofacial cleft patients during cleft correcting surgery (36 patients with unilateral cleft lip, 13 patients with bilateral cleft lip, 26 patients with isolated cleft palate). Control group oral cavity tissue was obtained from 7 patients without cleft lip and palate. To evaluate the number of immunopositive structures in the cleft affected tissue and the control group, a semiquantitative counting method was used. Non-parametric statistical methods (Kruskal–Wallis H test, Mann–Whitney U test, and Spearman’s rank correlation) were used. Results: Statistically significant differences for the number of PAX7, PAX9, and RYK-positive cells were notified between the controls and the patient groups. Multiple statistically significant correlations between the factors were found in each cleft affected tissue group. Conclusions: PAX7, PAX9, and RYK have a variable involvement and interaction in postnatal morphopathogenesis of orofacial clefts. PAX7 is more associated with the formation of unilateral cleft lip, while PAX9 relates more towards the isolated cleft palate. The stable presence of RYK in all cleft types indicates its possible participation in different facial cleft formations.

P76.71 RYK Confers Drug Tolerance to Osimertinib in Lung Cancer Cells with EGFR Mutations

Emergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance. Five EGFR-mutant NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array. DTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. Next, we compared the phosphorylation levels of EGFR in HCC4006 cells after exposure to 600 nM osimertinib or 60 nM afatinib. 600 nM osimertinib inhibited the phosphorylation of EGFR in HCC4006 cells more than 60 nM afatinib. This was inconsistent with the experiments using PC9 cells (both 600 nM osimertinib and 60 nM afatinib induced drug tolerance in PC9 cells), showing that inhibition of EGFR phosphorylation was similar between 600 nM osimertinib and 60 nM afatinib.To investigate if the suppression level of EGFR phosphorylation was related to the acquisition of a drug-tolerant phenotype, we exposed HCC4006 cells to different concentrations of osimertinib or afatinib. Lower doses of osimertinib (66 nM and 200 nM) failed to induce a drug-tolerant state in HCC4006 cells, although the growth inhibition rate of 66 nM osimertinib for HCC4006 parental cells was identical to that of 600 nM osimertinib. However, higher doses of afatinib (180 nM and 540 nM) induced a drug-tolerant state in HCC4006 cells. We also observed that lower doses of osimertinib and afatinib failed to induce a drug-tolerant state in PC9 and H1975 cells. These results indicated that stronger inhibition of phosphorylated EGFR is necessary to induce DTCs. Next, in the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. This phenomenon was HCC4006 cell line-specific since we observed that PC9 parental cells and PC9-600 nM osim-DTCs were both resistant to RYK knockdown. In addition, cabozantinib, which reportedly inhibit RYK, monotherapy effectively inhibited the proliferation of HCC4006-600 nM osim-DTCs but not HCC4006 parental cells. However, cabozantinib was not active against PC9-600 nM osim-DTCs and PC9 parental cells. Our study found that the inducibility of DTCs depended on the type of cell line and the drug concentration. It is possible that the optimal dose of EGFR-TKI in each patient may reduce the emergence of DTCs in clinical practice. We also found high expression of RYK was a molecular mechanism of the drug-tolerant state in HCC4006 cells against osimertinib. Further studies are necessary to fully understand the DTCs that are essential for the appropriate primary double-strike therapy for lung cancers with EGFR mutations.

Dose-dependence in acquisition of drug tolerant phenotype and high RYK expression as a mechanism of osimertinib tolerance in lung cancer

ObjectiveEmergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance. Materials and methodsFive EGFR-mutated NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array. ResultsDTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. We observed that a high drug concentration was required to induce DTCs, and HCC4006 cells become tolerant when a higher dose of afatinib (>180 nM) was used. In the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. ConclusionsThese results suggest that induction of DTCs is dose-dependent, and increased RYK expression was the mechanism of drug tolerance in HCC4006 cells against osimertinib.

Activity-dependent conformational transitions of the insulin receptor–related receptor

The insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor-related receptor (IRR) form a mini family of predimerized receptor-like tyrosine kinases. IR and IGF-1R bind to their peptide agonists triggering metabolic and cell growth responses. In contrast, IRR, despite sharing with them a strong sequence homology, has no peptide-like agonist but can be activated by mildly alkaline media. The spatial structure and activation mechanisms of IRR have not been established yet. The present work represents the first account of a structural analysis of a predimerized receptor-like tyrosine kinase by high-resolution atomic force microscopy in their basal and activated forms. Our data suggest that in neutral media, inactive IRR has two conformations, where one is symmetrical and highly similar to the inactive Λ/U-shape of IR and IGF-1R ectodomains, whereas the second is drop-like and asymmetrical resembling the IRR ectodomain in solution. We did not observe complexes of IRR intracellular catalytic domains of the inactive receptor forms. At pH 9.0, we detected two presumably active IRR conformations, Γ-shaped and T-shaped. Both of conformations demonstrated formation of the complex of their intracellular catalytic domains responsible for autophosphorylation. The existence of two active IRR forms correlates well with the previously described positive cooperativity of the IRR activation. In conclusion, our data provide structural insights into the molecular mechanisms of alkali-induced IRR activation under mild native conditions that could be valuable for interpretation of results of IR and IGF-IR structural studies.

RYK, a receptor of noncanonical Wnt ligand Wnt5a, is positively correlated with gastric cancer tumorigenesis and potential of liver metastasis.

Gastric cancer (GC) is the most prevalent human cancer around the globe. In GC, Wnt signaling is deregulated, and receptor-like tyrosine kinase (RYK) coreceptors have been identified to interact with noncanonical Wnt ligand Wnt5a. We, therefore, aimed to evaluate the role of RYK in GC development and metastasis. GC tumor samples were collected from 250 GC patients. Expressions of RYK, as well as markers for the epithelial-mesenchymal transition (EMT), such as N-cadherin and E-cadherin, were subjected to correlation analysis with clinicopathological features. Endogenous RYK expression levels were compared in GC cell lines with ascending metastatic potentials followed by stable RYK knockdown. Effect of RYK knockdown on GC cell migration, invasion, and EMT phenotype were assessed in vitro, and on GC tumor growth in vivo in a xenograft rodent model. Particularly, liver metastasis potential of tail vein-injected GC cells was also analyzed following RYK knockdown. RYK was highly correlated with liver metastasis of GC tumors and the expression profiles of EMT markers toward the mesenchymal tendency. RYK expression was also positively correlated with the metastasis potential of GC cells. RYK knockdown not only inhibited migration, invasion, and EMT of GC cells in vitro, but also suppressed tumorigenesis and liver metastasis of GC cells in vivo using the mouse xenograft model. RYK is highly correlated with GC tumorigenesis and potential of liver metastasis, suggesting it may be a novel oncogenic factor of the noncanonical Wnt signaling pathway contributing to GC.NEW & NOTEWORTHY RYK is highly correlated with gastric cancer tumorigenesis and the potential of liver metastasis, suggesting it may be a novel oncogenic factor of the noncanonical Wnt signaling pathway contributing to gastric cancer.

Choosing the right input in cell signaling

A decoding module discriminates ligands and signaling activation in the Wnt pathway

Insulin receptor‐related receptor as an extracellular pH sensor regulating bicarbonate excretion

The acid‐base balance in the organism at the system level is controlled primarily by the kidneys and lungs. In the kidneys, this function is performed by the intercalated cells of two types; alpha cells excrete excess acids while beta cells excrete excess bases as bicarbonate. In a result of the biochemical analysis of the insulin receptor‐related receptor (IRR), a receptor‐like tyrosine kinase enriched in beta‐intercalated cells, we discovered its ability to activate upon the extracellular treatment with mildly alkaline media. IRR is a close homolog of the receptors of insulin and insiln‐like growth factor but cannot be activated by to their agonists whereas IRR homologs do not respond to extracellular pH changes. This pH‐sensing property of IRR is evutionary conserved from amphibians to mammals. The IRR activation by alkali resembles a typical ligand‐receptor interaction since it is dose‐dependent and saturable, specific in relation to other homologous receptors, defined by the structure of the IRR ectodomain and accompanied by its conformational changes. Mutagenesis revealed two sites in the IRR ectodomain that act synergystically to induce the receptor activation. IRR activation triggers intracellular signaling resembling the insulin receptor pathway. We obtained a panel of monoclonal antibodies against the IRR ectodomain, one of them being able to activate the receptor, and another – to inhibit its response to alkaline media. Altogether, the obtained data suggest that IRR is a cellular pH sensor. We used kcnockout mice to address IRR systemic functions. The knockout mice are viable in normal conditions but fail to excrete excees bicarbonate upon experimentally induced alkalosis. Nevertheless, the increased blood bicarbonate is compensated by CO2 retention that keeps the blood pH within the physiological range. We performed comparative analyses of knockout and wild type mice kidney and brain transcriptomes and identified statistically valid changes in the expression of several proteins, in particular, ion transporters. Also, a behavioral phenotype of the knockout mice was revealed. They appeared to be less socially active and less agressive as compared to their wild‐type littermates.Support or Funding InformationThis work was financially supported by the Russian Science Foundation (grant № 14‐50‐00131)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Smek1/2 is a nuclear chaperone and cofactor for cleaved Wnt receptor Ryk, regulating cortical neurogenesis

The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.

RYK promotes the stemness of glioblastoma cells via the WNT/ β-catenin pathway.

Glioblastoma multiforme (GBM) is characterized by a strong self-renewal potential and a poor differentiation state. Since receptor-like tyrosine kinase (RYK) activates the WNT/β-catenin pathway essential for cancer stem cell maintenance, we evaluated its contribution in conferring stemness to GBM cells. Here, we report that Ryk (related-to-receptor tyrosine kinase), an atypical tyrosine kinase receptor, is upregulated in samples from GBM patients as well as in GSCs. Ryk overexpression confers stemness properties to GBM cells through the modulation of the canonical Wnt signaling and by promoting the activation of pluripotency-related transcription factor circuitry and neurosphere formation ability. In contrast, siRNA-mediated knockdown of Ryk expression suppresses this stem-like phenotype. Rescue experiments reveal that stemness-promoting activity of Ryk is attributable, at least in part, to β-catenin stabilization. Furthermore, Ryk overexpression improves cell motility and anchorage independent cell growth. Taken together, our findings demonstrate that Ryk promotes stem cell-like and tumorigenic features to glioma cells its essential for the maintenance of GSCs and could be a target of novel therapies.

Ryk receptors on unmyelinated nerve fibers mediate excitatory synaptic transmission and CCL2 release during neuropathic pain induced by peripheral nerve injury.

BackgroundNeuropathic pain is a major pathology of the central nervous system associated with neuroinflammation. Ryk (receptor-like tyrosine kinase) receptors act as repulsive axon-guidance molecules during development of central nervous system and neural injury. Increasing evidence suggests the potential involvement of Wnt/Ryk (wingless and Int) signaling in the pathogenesis of neuropathic pain. However, its underlying mechanism remains unknown.ResultsThe expression and location of Ryk receptor as well as its ligand Wnt1 were detected by qPCR, Western blot, and immunohistochemistry. We found that Ryk, a specific Wnt receptor, was expressed in IB4+ (Isolectin B4) and CGRP+ (calcitonin gene-related peptide) dorsal root ganglia neurons and their ascending unmyelinated fibers in the dorsal horn of the spinal cord. Ryk was upregulated after spinal nerve ligation surgery. Wnt1 was also increased in activated astrocytes in the dorsal horn after spinal nerve ligation. The presynaptic mechanism of Ryk in regulation of neuropathic pain was determined by electrophysiology in spinal slice. Spinal nerve ligation model was established, and the therapeutic potential of inhibiting Ryk receptor was determined. Spine-specific blocking of the Wnt/Ryk receptor signaling attenuated the spinal nerve ligation-induced mechanical allodynia but not thermal hyperalgesia. Further, it also blocked Ca2+-dependent signals including CaMKII and PKCγ, subsequent release of CCL2 (CCR-like protein) in the dorsal horn. An in vitro study showed that inactivating Ryk receptors with anti-Ryk antibodies or lentiviral Ryk shRNA led to the inactivation of Wnt1 for excitatory synaptic transmission in spinal slices and subsequent decrease in CCL2 expression in the dorsal root ganglia neurons.ConclusionThese studies demonstrate the existence of critical crosstalk between astrocytes and unmyelinated fibers, which indicate the presynaptic mechanism of Ryk in cytokine transmission of neuropathic pain and the therapeutic potential for Wnt/Ryk signaling pathway in the treatment of neuropathic pain.

Ryk is essential for Wnt-5a-dependent invasiveness in human glioma

Glioblastoma is characterized by marked invasiveness, but little is known about the mechanism of invasion in glioblastoma cells. Wnts are secreted ligands that regulate cell proliferation, differentiation, motility and fate at various developmental stages. In adults, misregulation of the Wnt pathway is associated with several diseases. Recently, we reported that Wnt-5a was overexpressed and correlated with cell motility and infiltrative activity through the regulation of matrix metalloproteinase (MMP)-2 in glioma-derived cells. Although several receptors for Wnt-5a were identified, the receptors of Wnt-5a that mediate cellular responses of glioma were not clearly identified. Knockdown of receptor-like tyrosine kinase (Ryk) but not that of Ror2 suppressed the activity of MMP-2 and Wnt-5a-dependent invasive activity in glioma cells. These results suggest that Ryk is important for the Wnt-5a-dependent induction of MMP-2 and invasive activity in glioma-derived cells and that Ryk might have a novel patho-physiological function in adult cancer invasion. Furthermore, not only the expression of Wnt-5a but also that of Frizzled (Fz)-2 and Ryk was correlated with the WHO histological grade in 38 human glioma tissues. Taking these findings together, Fz-2 and Ryk could be therapeutic or pharmacological target molecules for the control of Wnt-5a-dependent invasion of human glioma in the near future.

The Wnt Coreceptor Ryk Regulates Wnt/Planar Cell Polarity by Modulating the Degradation of the Core Planar Cell Polarity Component Vangl2

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2(+/-) background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a(-/-) mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.

P2-03-04: Novel Pathways Underlying the Initiation and Transition of DCIS to IDC of HER2−Overexpressing Breast Cancer Model.

Abstract Background: The HER2/Neu -oncogene is amplified in 20–25% percent of human primary breast cancers and this alteration is associated with disease behaviour. The HER2/neu oncogene encodes a receptor-like tyrosine kinase, whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. Whereas signalling pathways emanating from HER2 have been characterized, much less is known about the transcriptionally regulated genes controlled by HER2 that contribute to the initiation of Ductal Carcinoma Insitu (DCIS) and their transition to an invasive ductal carcinoma (IDC). Materials and Methods: Normal and HER2 overexpressing mammary epithelial cells (MCF10A) were grown in extracellular matrix to form 3D structures, When grown in extracellular matrix as 3-dimensional spheroids, control cells developed a hollow lumen, but HER2−overexpressing cells populated the lumen by evading apoptosis. On the next step, uopon the growth factror stimulation HER2−overexpressing cells grown in extracellular matrix, which protruded invasive outgrowths. Highly variable genes were selected from the described phenotypes using RNA isolated from the 3D structures and hybridized to an Affymetrix HuGene 1.0 ST oligonucleotide array. Results: Using microarrays we analysed transcriptional events responsible for the morphological changes and found several novel sets of genes such as integral proteins, transcription factors, matrix proteases, Notch genes and chemokines were highly altered in the HER2 overexpressing group which were not appreciated before. Using gene annotation we defined molecular-pathways responsible for the phenotypical changes. More specifically, our study proposes a two hit model describes the pathways involved in the initiation of the Ductal Carcinoma Insitu and their transition to Invasive Cancer underlying the HER2 transcriptional network. Discussion: According to the proposed model, expansion of ductal hyperplasia is limited by intraluminal apoptosis, unless they overexpress HER2, which drives proliferation and forms DCIS due to HER2 induced Notch pathways genes. Secondly, results obtained with the 3D system and their reflection in clinical outcome, we propose that neither HER2 amplification, nor the presence of GFs, is sufficient for development of IDC, but their co-occurrence can instigate metastasis by the activation of genes of integrin-adhesion signaling. The more virulent scenario combines HER2 amplification with GFs, thereby switching a robust, auto-stimulatory program. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-04.

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients' lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment.

Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.

Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling

Receptor-like tyrosine kinase (RYK) functions as a transmembrane receptor for the Wnt family of secreted protein ligands. Although RYK undergoes endocytosis in response to Wnt, the mechanisms that regulate its internalization and concomitant activation of Wnt signaling are unknown. We discovered that RYK both physically and functionally interacts with the E3 ubiquitin ligase Mindbomb 1 (MIB1). Overexpression of MIB1 promotes the ubiquitination of RYK and reduces its steady-state levels at the plasma membrane. Moreover, we show that MIB1 is sufficient to activate Wnt/β-catenin (CTNNB1) signaling and that this activity depends on endogenous RYK. Conversely, in loss-of-function studies, both RYK and MIB1 are required for Wnt-3A-mediated activation of CTNNB1. Finally, we identify the Caenorhabditis elegans orthologue of MIB1 and demonstrate a genetic interaction between ceMIB and lin-18/RYK in vulva development. These findings provide insights into the mechanisms of Wnt/RYK signaling and point to novel targets for the modulation of Wnt signaling.

His and Hers Receptors

Related receptor-like tyrosine kinases are differentially required in male and female gametophytes for fertilization.

T(3;21)(q22;q22) leading to truncation of the RYK gene in atypical chronic myeloid leukemia

The analysis of a small number of patients with atypical chronic myeloid leukemia showing balanced chromosomal translocations has revealed diverse tyrosine kinase fusion genes, most commonly involving FGFR1, PDGFRA, PDGFRB, JAK2, and ABL. We present a case of aCML with a 3q22;21q22-translocation that led to truncation of the receptor-like tyrosine kinase ( RYK) gene and its juxtaposition with sequences from chromosome 21 including the ATP5O gene coding for a mitochondrial ATP synthase. The resulting fusion was not in frame, however, which is why we speculate that an abrogated RYK gene product rather than a chimeric protein might be the leukemogenic result.